Chen et al. BMC Veterinary Research 2014, 10:24http://www.biomedcentral.com/1746-6148/10/24

RESEARCH ARTICLE Open Access

Sambucus nigra extracts inhibit infectiousbronchitis virus at an early point during replicationChristie Chen1, David M Zuckerman2, Susanna Brantley1, Michka Sharpe1, Kevin Childress1, Egbert Hoiczyk2

and Amanda R Pendleton1,3*

Abstract

Background: Infectious bronchitis virus (IBV) is a pathogenic chicken coronavirus. Currently, vaccination against IBVis only partially protective; therefore, better preventions and treatments are needed. Plants produce antimicrobialsecondary compounds, which may be a source for novel anti-viral drugs. Non-cytotoxic, crude ethanol extracts ofRhodiola rosea roots, Nigella sativa seeds, and Sambucus nigra fruit were tested for anti-IBV activity, since these safe,widely used plant tissues contain polyphenol derivatives that inhibit other viruses.

Results: Dose–response cytotoxicity curves on Vero cells using trypan blue staining determined the highestnon-cytotoxic concentrations of each plant extract. To screen for IBV inhibition, cells and virus were pretreated withextracts, followed by infection in the presence of extract. Viral cytopathic effect was assessed visually following anadditional 24 h incubation with extract. Cells and supernatants were harvested separately and virus titers werequantified by plaque assay. Variations of this screening protocol determined the effects of a number of shortenedS. nigra extract treatments. Finally, S. nigra extract-treated virions were visualized by transmission electronmicroscopy with negative staining.Virus titers from infected cells treated with R. rosea and N. sativa extracts were not substantially different frominfected cells treated with solvent alone. However, treatment with S. nigra extracts reduced virus titers by fourorders of magnitude at a multiplicity of infection (MOI) of 1 in a dose-responsive manner. Infection at a low MOIreduced viral titers by six orders of magnitude and pretreatment of virus was necessary, but not sufficient, for fullvirus inhibition. Electron microscopy of virions treated with S. nigra extract showed compromised envelopes andthe presence of membrane vesicles, which suggested a mechanism of action.

Conclusions: These results demonstrate that S. nigra extract can inhibit IBV at an early point in infection, probablyby rendering the virus non-infectious. They also suggest that future studies using S. nigra extract to treat or preventIBV or other coronaviruses are warranted.

Keywords: Infectious bronchitis virus, Coronavirus, Sambucus nigra, Nigella sativa, Rhodiola rosea

BackgroundAvian infectious bronchitis virus (IBV), a gamma-coronavirus, infects the respiratory tract of chickens andcauses the production of eggs with deformed and weak-ened shells [1,2]. The poultry and egg industries have con-sequently suffered large economic losses due to IBVinfections [3,4]. Current vaccination strategies target

* Correspondence: amanda.pendleton@actx.edu1Division of Natural Science and Mathematics, Oxford College of EmoryUniversity, Oxford, GA 30054, USA3Department of Mathematics, Sciences & Engineering, Amarillo College,Amarillo, TX 79178, USAFull list of author information is available at the end of the article

© 2014 Chen et al.; licensee BioMed Central LCommons Attribution License (http://creativecreproduction in any medium, provided the or

specific serotypes of the virus. However, vaccines have notproven wholly effective in protecting against new infectionsdue to the highly recombinant nature of the virus [5,6].More efficient methods of IBV prevention or treatment areclearly needed. Plant extracts may be a potential source ofagents for defending against IBV.Historically, plant extracts have been widely used to

treat various medical conditions [7-9]. Some of the best-known examples include quinine isolated from Cinchonapubescens (Cinchona tree) for treating malaria, digoxinfrom Digitalis purpurea (foxglove) for treating cardiacconditions, morphine from Papaver somniferum (opiumpoppy) used for pain, and aspirin synthesized from the

td. This is an open access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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bark of various Salix (willow) species. In many of thesecases, the active chemicals isolated from these plants havebeen the basis for developing additional medications thatare used today. Additionally, myriad plant extracts haveshown activity, both in vitro and in vivo, against a largerange of viral pathogens, including hepatitis B and C vi-ruses, herpes simplex virus, influenza virus, poliovirus,dengue viruses, and human immunodeficiency virus [10].Plant secondary metabolites, particularly polyphenols, arealso increasingly recognized as potent antimicrobials [11].In some cases this ability to use plant metabolites to com-bat animal pathogens may rise from the similarities inplant and animal innate immune systems [12]. Some com-monalities include the use of similar pathogen recognitionreceptors and MAP-kinase signaling pathways to upregu-late cellular immune responses, as well as reactive oxygenspecies and defensins to protect against invading mi-crobes. Therefore, it is not surprising that the secondarymetabolites used by plants for their own defense havebeen effective inhibitors, in some cases, of animal infec-tious agents [13]. One such secondary metabolite is cat-echin. In Picea abies (Norway spruce) and Carmelliasinensis (Chinese tea leaf), catechin-synthesizing genes areupregulated in response to fungal infection and are corre-lated with increased resistance to infection [14,15]. Inhumans, ingestion of or gargling with catechin-containingplant extracts results in lower rates of influenza virus infec-tion [16,17]. Quercetin is another secondary metabolite in-volved in plant and animal pathogen defense. Treatmentwith quercetin reduces susceptibility of Arabidopsis thaliana(mouse-ear cress) to Pseudomonas syringae infection [18].In vitro and in vivo studies have both shown that quercetinand its derivatives inhibit influenza virus and poliovirus rep-lication, while in vitro treatment of the human pathogen,Salmonella enterica, results in microbe death [19-24].The use of plant extracts as an alternative or supple-

mentary IBV treatment or prevention strategy has notbeen extensively investigated. The range of plants thathave been surveyed for their potential as anti-IBV agentsis also limited, although, purified compounds isolatedfrom Glycyrrhiza radix (licorice root) [25] and Forsythiasuspensa (weeping forsythia) [26] have shown effectivenessagainst IBV in vitro. However, the use of these extracts orthe active ingredients from these extracts for long-termtreatment or prevention strategies poses some toxicityconcerns [27-29]. These concerns, combined with the dif-ficulties often encountered when translating in vitro re-search into in vivo treatments [30], suggest that in vitroidentification of a number of different antiviral plants forfuture in vivo studies is important.This study investigated the effects of extracts of three

plant species – Rhodiola rosea (goldenroot), Nigella sativa(black cumin) and Sambucus nigra (common elderberry)– on avian IBV replication. To our knowledge, our study

is the first to test the effects of these plants on IBV replica-tion. We chose to study these plants due to their knownantiviral properties. For example, R. rosea extract hasshown antiviral activity against coxsackievirus B3 by pre-venting the virus from attaching and entering host cells[31]. R. rosea extracts also contain a number of antiviralchemicals, including gallic acid, caffeic acid, chlorogenicacid, and catechin [32], which have inhibited the replica-tion of human rhinoviruses [33], hepatitis B virus [34],and influenza virus [16,17]. N. sativa extract has shownantimicrobial properties against Escherichia coli, Bacillussubtilis, and other bacteria [35]. Studies of murine cyto-megalovirus infection and hepatitis C infection lend sup-port to the plant’s antiviral potential in vivo, as well[36,37]. Additionally, N. sativa compound extracts, espe-cially its saponins, alkaloids, and flavonols, show similaritieswith known antiviral chemicals [38-40]. Finally, S. nigra ex-tract has successfully inhibited influenza A and B virusesin vitro and in vivo [41]. S. nigra extracts are also character-ized by a high content of antiviral flavonoid anthocyanins[42]. Additionally, the antiviral compound quercetin islargely present in both S. nigra and in Amelanchier alnifolia(Saskatoon serviceberry) [43], a known inhibitor of the bo-vine coronavirus, in vitro [44]. Combined, these studies sug-gested that extracts of R. rosea, N. sativa, and S. nigra mightpossess broad antimicrobial or antiviral properties.Here we show that non-cytotoxic, crude ethanol extracts

of R. rosea roots and N. sativa seeds did not inhibit IBVinfection in vitro, while S. nigra fruit extracts inhibitedIBV by several orders of magnitude. This inhibition wasdose-responsive in that it decreased with decreasingS. nigra extract concentrations and increased with de-creasing virus concentrations. Treatment of virus with S.nigra extracts prior to infection was necessary, but not suf-ficient, for full virus inhibition. Additionally, electron mi-croscopy of virions treated with S. nigra extracts showedcompromised envelopes and the presence of membranevesicles. These results demonstrate that S. nigra extract caninhibit IBV at an early point in infection and suggest that itdoes so by compromising virion structure. Overall thesestudies identified a plant extract with previously unknowneffects against IBV, which could potentially lead to effectivetreatments or prevention of this or similar coronaviruses.

MethodsCells and virusesVero cells were maintained in high-glucose Dulbecco’smodified Eagle’s medium (DMEM) (Invitrogen Cor-poration, Carlsbad, CA) supplemented with 10% fetalcalf serum (Atlanta Biologicals, Norcross, GA) and0.1 mg/ml Normocin (Invivogen, San Diego, CA). Thepreviously described Vero-adapted Beaudette strain ofIBV [45] was used in all IBV infection experiments. In-fections and titers were performed with Vero cells.

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Preparation of plant extracts0.6 g of R. rosea powdered root (Starwest Botanicals Sacra-mento, CA) was incubated in 5 ml of 70% ethanol (Sigma-Aldrich, St. Louis, MO) for 24 h at room temperature[32]. 1.5 g of N. sativa seeds (Frontier Natural ProductsCo-op, Norway, IA) was homogenized in 10 ml of 85%ethanol and incubated for 7 d at room temperature[46,47]. 32.0 g of S. nigra fruit (San Francisco Herb Com-pany, San Francisco, CA) was homogenized in 40 ml of80% ethanol and incubated for 4 d at room temperature[48]. Following these incubations, extract solutions werecentrifuged at 1900 × g for 5 min at room temperature toremove debris and the remaining supernatant was syringefiltered through a 0.22 μm polyvinylidene fluoride mem-brane (Fisher Scientific Company, Fair Lawn, NJ). All ex-tract solutions were stored at 4°C.

Cytotoxicity assaysCells were plated in 35-mm dishes in duplicate for ap-proximately 1 d before being treated with plant extractsfor 48 h. Concentrations ranged from 7.5 × 10-5 g/ml to1.2 × 10-3 g/ml for R. rosea extract, from 9.4 × 10-5 g/mlto 1.5 × 10-3 g/ml for N. sativa extract, and from 5.0 ×10-4 g/ml to 8.0 × 10-3 g/ml for S. nigra extract. Thefinal concentration of solvent was kept constant in allwells at 0.04% ethanol for R. rosea extract treatments,0.2% ethanol for N. sativa extract treatments, and 0.4%ethanol for S. nigra extract treatments. At 48 h post-treatment, supernatants containing dead cells were col-lected and combined with adherent cells that had beenharvested using 0.05% trypsin (Invitrogen Corporation)in Dulbecco’s phosphate-buffered saline (Sigma-Aldrich,St. Louis, MO). 20 ml of this solution was then com-bined with an equal volume of 0.6% trypan blue (Sigma-Aldrich). The number of live cells per ml in each dishwas counted in duplicate using light microscopy and ahemocytometer. The relative cell viability was calcu-lated as live cells per ml in extract-treated dishes rela-tive to solvent-treated dishes.

Infection in the presence of plant extractsTo screen for anti-IBV effects, cells were plated in 35-mmdishes for approximately 2 d before being treated with3.75 × 10-4 g/ml of N. sativa extract, 1.5 × 10-4 g/ml of R.rosea extract, or 4.0 × 10-3 g/ml of S. nigra extract for24 h. Control cells for R. rosea, N. sativa, and S. nigra ex-tract treatments were incubated in final concentrations of0.04% ethanol, 0.2% ethanol and 0.4% ethanol, respect-ively. Prior to infection with IBV, virus was incubated withthese same concentrations of plant extract (or solventalone) for 20 min at room temperature. IBV infection wasthen performed at a multiplicity of infection (MOI) of ei-ther 1 or 0.1 by allowing virus to absorb to cells in a smallvolume of serum-free DMEM supplemented with plant

extract or solvent alone for 1 h at 37°C. Cells were thentransferred to fresh DMEM supplemented with 10% fetalcalf serum, antibiotics, and plant extract or solvent for anadditional 24 h. Viral cytopathic effect was then assessedvisually using light microscopy, and virus-infected super-natants and cells were harvested separately. Supernatantswere collected, centrifuged at 1900 × g for 5 min at roomtemperature to remove cellular debris, and stored at −80°Cuntil virus titers could be determined. Cells were trans-ferred to fresh DMEM supplemented with 10% fetal calfserum and antibiotics before being lysed by three roundsof freeze-thaw. Centrifugation at 1900 × g for 5 min atroom temperature removed cellular debris and theremaining supernatant was stored at −80°C until virus ti-ters could be determined.After initial screening, the following S. nigra extract

treatments were assessed for their ability to inhibit IBVeither alone or in combination: (1) exposing cells to ex-tract prior to infection, (2) exposing cells to extract fol-lowing infection, (3) exposing virus to extract prior toinfection, (4) exposing both cells and virus to extractduring infection. Infections were done at an MOI of 0.1as indicated above, except that exposure to solvent alonewas substituted for exposure to S. nigra extract if a spe-cific treatment was omitted. For example, to determinethe effects of only exposing cells to S. nigra extract priorto infection, cells were first incubated with 4 mg/ml of S.nigra extract for 24 h prior to infection. Virus was thenincubated in solvent alone for 20 min prior to infectionand solvent was present during infection. Cells werethen incubated in solvent alone for an additional 24 hfollowing infection before being harvested, as describedabove.

Plaque assaysVirus titers were quantified via plaque assay. First, serial di-lutions of virus were absorbed to confluent Vero cells for1 h in a small amount of serum free DMEM. Virus wasthen removed from cells and an agarose overlay (equal vol-umes of 2× DMEM and 1.8% melted agarose (InvitrogenCorporation)) was added. After 2 d, an additional agaroseoverlay containing 0.015% neutral red (MP Biomedicals,LLC, Solon, OH) was added to cells. Approximately 24 hlater, clear plaques were counted and virus titers were cal-culated in particle forming units/ml (pfu/ml).

Electron microscopyTo purify virus, 30 ml of cell culture supernatant wasoverlaid on 4 ml of 20% sucrose in TNE buffer (50 mMTris, pH 7.4, 100 mM NaCl, 1 mM EDTA) and 2 ml of55% sucrose in TNE in an SW 28 tube (Beckman-Coulter,Brea, CA). Samples were spun for 3 h at 25 k RPM in anSW 28 rotor. Purified virus was collected from the 20%-55% sucrose interface, diluted with TNE and pelleted for

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2 h at 55 k RPM in an SW 55Ti rotor. Pellets were re-suspended in 40–60 μl TNE and kept on ice for imme-diate use.Purified virus was treated with 8.0 × 10-3 g/ml of S. nigra

extract or 0.8% ethanol as a vehicle control in PBS for15 min at room temperature. Samples were then spottedonto a glow discharged, carbon coated copper grid (Elec-tron Microscopy Sciences, Hatfield, PA) and incubated for2 min. Grids were rinsed with water, and stained for 1 minwith 2% phosphotungstinic acid, pH 7.4. Samples were ex-amined on a Hitachi 7600 transmission electron micro-scope under 80 kV, and micrographs collected using AMTImage Capture Engine software controlling an AMT ER505 megapixel CCD camera (Advanced Microscopy Tech-niques Corp., Danvers, MA).

Ethical approvalThe research protocol used for this study was approved bythe Health & Biosafety Committee at Emory University(Biosafety File #: 08-2528-11). No human or animal sub-jects were used.

ResultsDetermining non-cytotoxic concentrations ofplant extractsScreening of plant extracts for antiviral potential must bedone using non-cytotoxic concentrations of extract.Therefore, cytotoxicity assays with trypan blue stainingwere performed. Cells were treated for 48 h with the indi-cated concentration of N. sativa, R. rosea, or S. nigra ex-tracts and the number of live cells for each concentrationof extract, relative to solvent treatment alone, was deter-mined. For all plant extracts, the number of live cells de-creased with increasing concentrations of extract in adose-responsive manner (Figure 1). The highest concen-tration of plant extract that did not significantly decreasethe number of live cells, relative to controls, was used forall subsequent antiviral screening.

N. sativa and R. rosea extracts do not inhibit IBV, whileS. nigra extracts doAntiviral agents may exhibit an effect via myriad mecha-nisms. Therefore, screening was performed using extractbefore, during, and after infection to maximize the pos-sibility of detecting antiviral action. Cells were treatedfor 24 h prior to infection with the indicated concentra-tion of extract. Virus was treated for 20 min prior toinfection and extract was present during the 1 habsorption of virus to cells. Cells were then treated foran additional 24 h post-infection (p.i.). Treatment withsolvent alone was used as a control. At 24 h p.i. cellswere visually assessed for viral cytopathic effect (CPE).Supernatants and cells were harvested separately andviral titers were quantified.

Virus titers of the N. sativa extract-treated superna-tants and cells were not significantly different from con-trols (Figure 2A). Unexpectedly, R. rosea extract-treatedsupernatants and cells showed a small, yet reproducible,two-fold increase in virus titers (Figure 2B). On theother hand, S. nigra extract-treated cells showed no de-tectable CPE at an MOI of 0.1 and a reduction of virustiters by six orders of magnitude (Figures 2C & 2D). In-hibition was not as great in S. nigra extract-treated sam-ples when a higher MOI of 1 was used (Figure 2E).However, this inhibition was still large, reducing viral ti-ters by approximately four orders of magnitude, relativeto solvent-treated samples. Virus titers also decreasedwith increasing S. nigra extract concentrations in a dose-responsive manner, indicating that S. nigra extract treat-ment was responsible for virus inhibition.

S. nigra extracts inhibit IBV at an early step in theinfection processTo begin uncovering the mechanism by which S. nigraextracts inhibited IBV, we assessed the impact of short-ened S. nigra extract treatments on IBV replication. Aseries of infections were done in which only cells weretreated with extract prior to infection (pre-C), only viruswas treated prior to infection (pre-V), or only treatmentfollowing infection was done (post). The pre-C treat-ment did not result in any reduction in virus titer rela-tive to treatment with solvent alone (Figure 3). Similarly,virus titers were not reduced in the cells of samples thatreceived only the post treatment. However, the posttreatment did result in a modest, three-fold reduction intiters of the supernatants. On the other hand, the pre-Vtreatment resulted in a titer reduction of over three or-ders of magnitude in the cells and over four orders ofmagnitude in the supernatants. Clearly, out of the threeshortened treatments tested, the pre-V treatment aloneshowed the greatest inhibition. However, this treatmentwas not sufficient for reducing virus titers to the samelevel as when all three treatments were combined.To further explore the effects and potential synergy of

different timings of extract exposure, another series of in-fections was done with varying extract treatment scenarios,as indicated (Figure 4). Results from these experiments re-vealed that combining pre-V treatment with post treatmentworked together to fully inhibit IBV replication. The pre-Ctreatment was not necessary for full virus inhibition, nordid it impact the viral titer of the supernatant. However, itdid work synergistically with pre-V treatment to reduceviral titers in the cells an additional three orders of magni-tude, as compared to pre-V treatment alone. In addition,exposing virus to S. nigra extract at the time of infectiondid not reduce virus titers, unless it was combined with thepost treatment. In every combination of treatments, pre-treating the virus with S. nigra extract greatly increased

Figure 1 Determining non-cytotoxic concentrations of each plant extract. Vero cells were treated for 48 hours with the indicatedconcentration of plant extract. Independent cytotoxicity assays were performed three times, with four replicates per assay, using trypan blue staining.Error bars represent standard deviation. Starred data points represent the highest concentration of extract that was not significantly different from thecontrol by a student’s t-test (p > 0.05). These starred concentrations were used in all subsequent infection assays, unless noted otherwise.

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virus inhibition. Finally, combining the pre-C and posttreatments did result in a further two orders of magnitudetiter reduction in the supernatants and cells, when com-pared to post treatment alone. Taken together these resultsindicate that some treatments worked together to fully in-hibit IBV replication. Importantly, the necessity and largeeffect seen with pre-V treatment indicated that one mech-anism of inhibition occurs at an early step of the IBV repli-cation cycle.

S. nigra extract compromises IBV virion structureTo explore if the extracellular effect of S. nigra extract onIBV infectivity was due to physical disruption of the virion,virus samples treated with S. nigra extract or solvent alonewere negative stained and examined by transmission elec-tron microscopy. Intact virions with uncompromised enve-lopes and characteristic spike protein profiles were easilyidentified in solvent treated samples (Figure 5A). By con-trast, treatment of the virus with S. nigra extract resulted

Figure 2 N. sativa and R. rosea extracts do not inhibit IBV, while S. nigra extracts do. A – D) Cells were pretreated for 24 h and virus for20 min with 3.75 × 10-4 g/ml N. sativa extract, 1.5 × 10-4 g/ml R. rosea extract, 4.0 x 10-3 g/ml S. nigra extract, or solvent alone prior to infection inthe presence of extract. The same concentration of extract was also present during virus absorption to cells. Cells were then treated for anadditional 24 h p.i. with the same concentration of extract. Independent infections with IBV were performed three times at an MOI of 0.1, withtwo replicates per assay. E) Cells were treated as for A – D, except that different concentrations of S. nigra extract were used, as indicated.Additionally, IBV infections were performed at an MOI of 1. A, B, C, E) Quantitation of virus titers at 24 h p.i. was done by plaquing in duplicateusing neutral red staining. D) Visualization of viral CPE was done at 24 h p.i. via light microscopy. A) N. sativa, B) R. rosea, C – E) S. nigra.

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Figure 3 Pre-treatment of IBV with S. nigra extracts dramatically reduces viral titers. Cells and virus were treated with 4.0 x 10-3 g/ml ofS. nigra extract as indicated below. Infection was done at an MOI of 0.1. Quantitation of virus titers at 24 h p.i. was done by plaquing in duplicateusing neutral red staining. Independent infections with IBV were performed three times, with two replicates per assay.

Figure 4 Treating IBV with S. nigra extracts prior to infection is necessary for full virus inhibition and works synergistically withtreating cells after infection. Cells and virus were treated with 4.0 x 10-3 g/ml of S. nigra extract as indicated below. Infection was done at anMOI of 0.1. Quantitation of virus titers at 24 h p.i. was done by plaquing in duplicate using neutral red staining. Independent infections with IBVwere performed three times, with two replicates per assay.

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Figure 5 Treating IBV with S. nigra extracts compromises virion structure. Virus was treated with 8.0 x 10-3 g/ml of S. nigra extract orsolvent (vehicle) alone for 10 min before being prepared for transmission electron microscopy with negative staining. A) Most frequent virionstructures observed. bar = 100 nm. Arrows indicate vesicle structures. B) Aggregates of vesicle structures observed only in virus samples treatedwith S. nigra. bar = 100 nm.

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exclusively in virions with damaged envelopes. The profilesof the spike proteins appeared unaffected in the treatedsamples, but the membranous envelope appeared to havebeen compromised (Figure 5A). Additionally, in the extracttreated samples, many spheres, resembling membrane vesi-cles, were seen surrounding the virions (Figure 5A) or clus-tered together in large aggregates (Figure 5B). Thesevesicles were of relatively uniform size (24.4 +/− 1.7 nm, n= 58) and were only apparent in extract treated virions,and not in solvent treated virus, or extract alone (Figure 5and data not shown). Taken together, these data indicatethat the pre-treatment of IBV with S. nigra extract resultsin extensive membrane damage to the virus, likely render-ing it non-infectious.

DiscussionVaccination against IBV, a pathogen that causes largeeconomic losses among the egg and poultry industries,has not proven wholly effective; therefore, alternativetreatment or prevention strategies are needed. Here wescreened non-cytotoxic (Figure 1), crude ethanol extractsfrom S. nigra berries, N. sativa seeds, and R. rosea rootsfor antiviral effects. Only S. nigra extracts inhibited viralreplication, reducing viral titers by four to six orders ofmagnitude in a dose-dependent manner (Figure 2). S.nigra extract treatment of only virus prior to infectiondrastically inhibited the virus (Figure 3), indicating thatS. nigra extract inhibits IBV at an early point in the in-fection process. Electron microscopy of S. nigra extract-treated IBV revealed compromised virion structures andmembranous vesicles (Figure 5), which were not presentin the extract alone. Therefore, S. nigra extract disruptsIBV virion structure, likely rendering it non-infectious.Our results raise questions about which compounds

within the crude S. nigra extract inhibit IBV, as well astheir mechanisms of action. Polyphenols are a likelysource of this inhibition, as plants with high polyphenolconcentrations often have antiviral properties [11]. Infact, two flavonols extracted from S. nigra berries canbind to virions from specific influenza virus strains andprevent infection in vitro [48], although whether these

flavonols disrupted virion structure is unknown. Perhapsthese or similar compounds in our S. nigra extract alsoinhibited IBV. Intriguingly, S. nigra extract has now beenshown to inactivate two enveloped viruses, in the case ofIBV by compromising its membrane directly. The mem-branes of these two viruses are chemically distinct, withIBV membranes being derived from the endoplasmicreticulum Golgi intermediate compartment, while influ-enza membranes are derived from the plasma membrane.These results suggest that S. nigra extract may have broadanti-viral effects against other enveloped viruses.In addition to polyphenols, lectins are commonly found

in plant extracts and often show antiviral activity by bindingto viral proteins or host receptors, preventing their inter-action [49-54]. S. nigra berry extracts are known to containthree plant lectins [55-59]. Two of these lectins possess spe-cificity for galactose and N-acetylgalactosamine, while theother one preferentially binds α2,6-linked sialic acid. Al-though IBV, a gamma-coronavirus, depends upon sialylatedhost receptors for entry into cells, it specifically uses α2,3-linked moieties, not α2,6-linked moieties [60]. Therefore, itis unlikely that S.nigra lectins block access to host-cell re-ceptors used by IBV. Our results support this idea, sincetreatment of cells prior to infection had no effect on viralreplication (Figure 3). On the other hand, IBV proteins,such as the spike protein, contain several consensus se-quences that signal the addition of N-linked oligosaccha-rides [61]. Possibly, S. nigra lectins could bind directly toviral proteins and inhibit infection. Lectins bound to the vi-rions of both an alpha- and beta-coronavirus did inhibit in-fection [62], lending support to this idea. How binding by S.nigra lectins and virion disruption (Figure 5) would be re-lated is unclear and might occur by separate mechanisms.While N. sativa and R. rosea extracts did not inhibit

IBV, many of their phytochemicals are thought to be anti-viral. For example, N. sativa seed extracts predominantlycontain saponins, glycosides, terpenoids and alkaloids[38,63-67], many of which are similar to known antiviralchemicals [38-40,68]. On the other hand, R. rosea root ex-tracts consist of many kaempferol, herbacetin, dihydro-myricetin, and myricetin derivatives [32]. Of these R. rosea

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compounds, kaempferol, gossypetin, and salidroside haveshown strong antiviral effects against influenza and Cox-sackie viruses [69,70]. S. nigra is also rich in cyanidin,kaempferol, myricetin, dihydromyricetin, and quercetin de-rivatives [42,71,72], making it much more similar chem-ically to R. rosea than to N. sativa. However, chemicals thatare found in S. nigra berry extracts, but not in either R.rosea or N. sativa extracts, are particularly attractive candi-dates for future tests into the chemical nature of S. nigraextract inhibition. These S. nigra chemicals include severalcyanidin derivatives; 3-, 4-, and 5-caffeoylquinic acid;kaempferol 3-rutin; rutin; pelargonidin 3-glucoside; iso-rhamnetin 3-rutin; and isorhamnetin 3-glucoside. Cyani-dins, kaempferols, and isorhamnetins are known antiviralchemicals [68]. Additionally, the two flavonols (5,7,3’,4’-tetra-O-methylquercetin and 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexa-necarboxylate), which bind to and inhibit influenza virus[48], are found in S. nigra and not in R. rosea or N. sativa,making them potential candidates as well. Alternatively,testing different fractions of S. nigra extracts for antiviralcapabilities, along with direct chemical identification, couldidentify which, if any, of these chemicals are responsible forthe early inhibition of IBV replication. In addition, otherplant extracts with chemicals that are similar to those in S.nigra extracts might also be considered for future anti-IBVtests. For example, extracts from A. alnifolia berries,branches, and leaves have chemicals (3-carreolyquinic acidand cyanidin 3-glucoside) that are found in S. nigra but notin R. rosea or N. sativa [42,71,73]. And indeed, A. alnifoliabranch extracts inhibited the bovine coronavirus in vitro[44]. Finally, a currently unidentified chemical or combin-ation of chemicals may be responsible for the ability of S.nigra extract to compromise IBV virion structure. One pos-sibility may be cholesterol chelators, since they are known tocompromise the membrane integrity of other viruses, result-ing in a loss of infectivity [74]. Currently, none of the chemi-cals known to be present in S. nigra berry extracts chelatecholesterol or have vesiculating effects on lipid membranes;however, future studies may demonstrate otherwise.Various combinations of S. nigra extract treatments also

showed synergistic inhibition. For example, complete in-hibition occurred when pre-treatment of virus was donein combination with post-infection treatment (Figure 4).Potentially, this synergy is due solely to compromised vir-ion structure, since these experiments were done at a lowMOI and allowed more than one round of replication tooccur. Specifically, virions that survive the pre-treatmentintact would be competent for infection, and their progenywould face no further challenge from the extract in the ab-sence of post-infection treatment. Alternatively, the syner-gistic inhibition of infected cells seen when pre-treatmentof virus and pre-treatment of cells were combined may in-dicate that more than one mechanism is at work and that

more than one active compound is present in the crudeextract. Again, testing of S. nigra extract fractions will helpexplore this possibility.If polyphenols in S. nigra extract are the cause of inhib-

ition, growing conditions and cultivars could greatly affectthe antiviral properties of the plant extracts. For example,the Korsør, Haschberg, and Rubini cultivars of S. nigra varyin their phenolic concentrations [42,71,75]. In addition,within each cultivar of S. nigra, the polyphenols varythroughout different growing seasons [71]. If in vivo testsalso demonstrate IBV inhibition by S. nigra extract, identi-fying the most efficient cultivar and growing conditions forS. nigra may be important for any practical treatment orprophylactic applications of this research.Additionally, it should be noted that the attenuated

Beaudette strain was used for all experiments presented inthis paper. In vitro screening using the Beaudette strain hasled to the identification of virucidal botanicals that were ef-fective in chicken populations [76]. Therefore, some prece-dence exists for successful prediction of in vivo efficacyusing this attenuated strain. This success may be due, inpart, to the high amino acid identity (96.3%) between thespike proteins of the Beaudette strain and the highly patho-genic Massachusetts M41 strain of IBV [61]. Experimentsusing S. nigra in chicken populations infected with virulentstrains will be important for directly assessing the in vivopotential of this plant extract.Interestingly, while vaccination is the main method for

inhibiting IBV in poultry populations [77], its effectivenesson new strains is often minimal, leading to outbreaks ineven vaccinated populations [78-80]. Perhaps vaccination inconjunction with administering the active polyphenol couldhave a synergistic effect, similar to that seen when the poly-phenol isoquercetin was administered with the influenzamedicine amantadine in vitro [24]. Finally, these resultshave the potential to translate into treatments for other cor-onaviruses, including those that affect humans. These hu-man coronaviruses (HCoV) include ones that may cause upto 20% of the common cold (HCoV 229E, HCoV OC43);HCoV NL63 and HCoV HKU1, which cause mild to severerespiratory diseases; the SARS CoV, which emerged in 2003with a 10% mortality rate; and the recently emerged MERSCoV, which currently has a 57% case fatality rate [81,82].Some evidence supports this idea, in that glycyrrhizin, theactive chemical from G. radix extracts, inhibited not onlyIBV, but also the SARS CoV [25,83].

ConclusionsTaken together, our studies have identified a plant ex-tract from Sambucus nigra with previously unknowninhibitory effects against IBV. We have also identifiedthe likely mechanism of this inhibition. Our resultscould potentially lead to effective treatments or pre-vention of IBV or similar coronaviruses.

Chen et al. BMC Veterinary Research 2014, 10:24 Page 10 of 12http://www.biomedcentral.com/1746-6148/10/24

AbbreviationsIBV: Infectious bronchitis virus; CPE: Cytopathic effect; MOI: Multiplicity ofinfection; p.i.: Post-infection; pre-C: Treatment of cells with plant extract for 24 hprior to infection; pre-V: Treatment of virus with plant extract for 20 min prior toinfection; post: Treatment with plant extract for 24 h following infection.

Competing interestsNo financial or non-financial competing interests exist for any of the authorsof this study.

Authors’ contributionsCC participated in the design of the S. nigra portion of the study, carried outmost of the S. nigra experiments, drafted substantial portions of themanuscript, and helped prepare the final manuscript. DMZ participated inthe design of the transmission electron microscope experiments, carried outthese experiments, drafted a portion of the manuscript, and helped preparethe final manuscript. SB participated in the design of the N. sativa portion ofthe study, carried out all of the N. sativa experiments and some of theR. rosea experiments, and helped prepare the final manuscript. MSparticipated in the design of the R. rosea portion of the study, carried outsome of the R. rosea experiments, and helped prepare the final manuscript.KC drafted a substantial portion of the manuscript and helped prepare thefinal manuscript. EH participated in the design of the transmission electronmicroscope experiments and helped prepare the final manuscript. ARPconceived of the study, participated in the design of the study, carried outsome of the S. nigra experiments, coordinated all authors’ efforts, draftedportions of the manuscript, and helped prepare the final manuscript. Allauthors read and approved the final manuscript.

AcknowledgementsWe would like to thank Dr. Carolyn Machamer of The Johns HopkinsUniversity School of Medicine for providing us with virus and cells. Thisresearch was made possible by funding from the Howard Hughes MedicalInstitute (Grant No. 52005873), the Summer Undergraduate Research atEmory program, the Pierce Institute for Leadership and CommunityEngagement at Oxford College of Emory University, the Harrison FoundationEquipment Grant, and the President’s Supplemental Fund for Scholarship atEmory University. DMZ. and EH were supported by a grant from the NationalInstitutes of Health (GM85024).

Author details1Division of Natural Science and Mathematics, Oxford College of EmoryUniversity, Oxford, GA 30054, USA. 2W. Harry Feinstone Department ofMolecular Microbiology and Immunology, The Johns Hopkins UniversitySchool of Public Health, Baltimore, MD 21205, USA. 3Department ofMathematics, Sciences & Engineering, Amarillo College, Amarillo, TX 79178,USA.

Received: 12 July 2013 Accepted: 10 January 2014Published: 16 January 2014

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doi:10.1186/1746-6148-10-24Cite this article as: Chen et al.: Sambucus nigra extracts inhibit infectiousbronchitis virus at an early point during replication. BMC Veterinary Research2014 10:24.

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